Attenuated polioviruses

ABSTRACT

An attenuated enterovirus or rhinovirus, suitable for use as a vaccine, has an attenuating mutation at least at a position which is, or corresponds with, position 479 and/or 482 of poliovirus type 3 Leon strain.

This is a continuation of application Ser. No. 07/543,759, filed Jul.19, 1990, now abandoned.

This invention relates to construction of vaccines against rhinovirusesand enteroviruses, particularly polioviruses, by the introduction ofdefined mutations into their genomes. These mutations attenuate thevirulence of wild type viruses and can further attenuate existing liveattenuated vaccine virus strains, thereby making them less likely torevert to virulence.

At the present time, the only vaccines routinely used againstenterovirus and rhinovirus infections are those against poliomyelitis.Of these the live attenuated vaccines developed by Sabin in the 1950'shave found greatest use throughout the world. Vaccine strains derivedfrom each of the three poliovirus stereotype (P1, P2 and P3) wereprepared by passage of wild type viruses in cell cultures and wholeanimals until attenuated strains were obtained. These attenuated virusesare substantially less able to cause poliomyelitis in humans than theoriginal wild type strains. They are administered orally and replicatein the gut to induce a protective immune response.

Although these vaccines are generally regarded as safe, their use isassociated with a low level of paralysis in vaccinees. This is mostoften associated with type 2 and type 3 serotypes and rarely, if ever,with type 1. There is therefore a requirement for improved type 2 andtype 3 vaccines which would be comparable in safety to the excellenttype 1 strain. There is also a requirement for vaccines against otherenteroviruses, e.g. echo, coxsackie and hepatitis A, and againstrhinoviruses.

The Sabin vaccine strains were developed by essentially empiricalprocedures. The genetic basis of their attenuation is not properlyunderstood. Over the past few years, however, scientists have employed anumber of molecular biological techniques in an attempt to elucidate themechanism by which the neurovirulence of these vaccine strains isreduced. Most of the work has concentrated on serotypes 1 and 3. Forboth of these the complete nucleotide sequences of the vaccine strainshave been compared with those of their neurovirulent progenitors.

In the case of poliovirus type 1, the vaccine strain differs from itsprogenitor at 47 positions in the 7441 base genome (Nomoto et al, 1982,Proc Natl Acad Sci U.S.A. 79: 5793-5797). All of these are simple pointmutations and 21 of them give rise to amino acid changes in virus codedproteins. Although several mutations are thought to contribute to theattenuation phenotype of the vaccine strain, direct evidence has beenpresented that the mutation of A to G at position 480 in the 5'non-coding region of the genome has a marked attenuating effect on thevirus (Nomoto et al, 1987, UCLA Symp Mol Cell Biol, New Series, Vol 54(Eds M A Brinton and R R Rueckert), 437-452, New York: Alan R Liss Inc).

Analogous studies on poliovirus type 3 reveal just 10 nucleotidesequence differences in the 7432 base genome between the vaccine and itsprogenitor strain (Stanway et al, 1984, Proc Natl Acad Sci U.S.A. 81:1539-1543). Just three of these give rise to amino acid substitutions invirus encoded proteins. The construction of defined recombinants betweenthe vaccine and its progenitor strain has allowed the identification ofthe mutations which contribute to the attenuation phenotype. One ofthese is at position 2034 and causes a serine to phenylalanine change invirus protein VP3.

The other mutation of interest is C to U at position 472 in the 5'non-coding region of the genome. This latter mutation has been observedto revert to the wild type C rapidly upon replication of the virus inthe human gut (Evans et al, 1985, Nature 324: 548-550). This reversionis associated with an increase in neurovirulence. C at position 472 hasalso been shown to be essential for growth of a mouse/human poliorecombinant virus in the mouse brain (La Monica et al, 1986, J Virol 57:515-525). Recently, we have observed that at 481 in poliovirus type 2 Achanges to G in an analogous fashion upon replication of the type 2vaccine in the gut of vaccinees.

We have investigated mutations of the wild-type poliovirus type 3 Leonstrain at several sites in the 5' non-coding region approximatelyspanning nucleotides 450 to 510. We found that poliovirus with amutation at position 479 or 482 is attenuated but with a mutation atposition 480 is non-attenuating. Multiple mutations were frequentlylethal. However, Poliovirus with a double mutation at positions 472 and482 was more highly attenuated than poliovirus with a single mutation ateither position.

The findings can be extrapolated to all polioviruses. indeed, they maybe extrapolated to other enteroviruses and rhinoviruses. Mutations atsites of other-enteroviruses and rhinoviruses corresponding to position479 and/or 482 and, optionally, position 472 of poliovirus type 3 Leonstrain can lead to attenuation. There is a relatively high degree ofhomology between the genome RNA of all enteroviruses and rhinoviruses.The positions of another strain of enterovirus or rhinoviruscorresponding to positions 472, 479 and 482 of poliovirus type 3 Leonstrain (based on the numbering used in the Stanway et al paper alreadyreferred to) can be determined by lining up the sequences of the genomicRNA of the strains.

Accordingly the invention relates to attenuated enteroviruses andrhinoviruses having an attenuating mutation at least at a position whichis, or corresponds with, position 479.and/or 482, and optionally alsoposition 472, of the genome of poliovirus type 3 Leon strain.

The present invention is particularly applicable to polioviruses. Wehave found in particular that the mutation A to C at position 479 of thegenome of poliovirus type 3 Leon strain causes attenuation, as doesnutation G to A at position 482. Either or both may be combined with themutation C to U at position 472. We have further found that poliovirustype 3 with mutations at both positions 472 and 482 is more highlyattenuated than viruses with mutations at either one of the positions.

An attenuated poliovirus may be a type 1, type 2 or type 3 poliovirus.Types 2 and 3 are preferred. For types I and 2, positions 476, 479 and469 correspond to positions 479, 482 and 472 respectively of poliovirustype 3. An attenuated type 1 or type 2 poliovirus therefore includes anattenuating mutation at position 476 and/or 479 and, optionally, also atposition 469. These mutations may be as above for type 3.

An attenuated virus according to the invention is prepared by a processcomprising:

(i) introducing the or each desired mutation by site-directedmutagenesis into a sub-cloned region, which includes the or eachposition it is wished to mutate, of a cDNA copy of the genome of anenterovirus or rhinovirus;

(ii) reintroducing the thus modified region into the complete cDNA fromwhich the region was derived; and

(iii) obtaining live virus from the cDNA thus obtained.

A mutation can be introduced into a strain of an enterovirus orrhinovirus, for example wild-type virus, by site-directed mutagenesis ofa cDNA copy of its genomic RNA. This may be achieved beginning withsub-cloning the appropriate region from an infectious DNA copy of thegenome of any of the virus strain, for example a vaccine strain or itsprogenitor, into the single strand DNA of a bacteriophage such as M13.The virus strain may be a neurovirulent strain but is preferably avaccine strain. For poliovirus it may be a Sabin, type 3 Leon or type 1Mahoney strain. The or each desired nutation is then introduced intothis sub-cloned cDNA using the technique of oligonucleotide directedmutagenesis.

After the introduction of mutations, the modified sub-cloned cDNAs arereintroduced into the complete cDNA from which they were derived and,for virulence testing in mice, into a cDNA derived from a murinepoliovirus derivative known to cause a poliomyelitis type disease inmice (La Monica et al). Live virus is recovered from the mutated fulllength cDNA by production of a positive sense RNA typically using a T7promoter to direct transcription in vitro (Van der Werf et al, 1986,Proc Natl Acad Sci, U.S.A. 83:2330-2334). The recovered RNA may beapplied to tissue cultures using standard techniques (Koch, 1973, CurrTop Microbiol Immunol 61:89-138). After 4-6 days incubation virus can berecovered from the supernatant of the tissue culture. The level ofneurovirulence of the modified virus may then be compared with that ofthe unmodified virus using a standard LD50 test in mice (La Monica etal) or the WHO approved vaccine safety test in monkeys (WHO Tech Rep Ser687: 107-175, 1983).

The attenuated viruses can be used as vaccines. They may therefore beformulated as pharmaceutical compositions further comprising apharmaceutically acceptable carrier or diluent. Any carrier or diluentconventionally used in vaccine preparations may be employed. Forexample, the presently used live attenuated poliovirus strains arestabilised in a solution of 1 molar MgCl₂ ana administered as a mixtureof the three serotypes.

The attenuated viruses can therefore be used to prevent an infectionattributable to an enterovirus or rhinovirus in a human patient. Forthis purpose, they may be administered orally, as a nasal spray, orparenterally, for example by subcutaneous or intramuscular injection. Adose corresponding to the amount administered for a conventional livevirus vaccine, such as up to 10⁶ TCID₅₀ for a Sabin vaccine strain inthe case of poliovirus, may be administered.

The following Example illustrates the invention.

EXAMPLE

A HindIII-Sstl fragment from a cDNA clone of P3/Leon/37, which includesthe first 751 base pairs of the genome, was sub-cloned into the phagevector M13mp9. P3/Leon/37 is the neurovirulent progenitor of the type 3vaccine strain. Mutations were then introduced into this sub-cloned cDNAfragment using the technique of oligonucleotide directed mutagenesis.The chemically synthesised DNA oligonucleotides used are shown in Table1 below:

                  TABLE 1                                                         ______________________________________                                        Oligo-                                                                        nucleo-                                                                       tide No                                                                             Sequence (5'-3')                                                        ______________________________________                                        1     GCC-TGC-TCC-ATG-GTT-ATA-TTT-AGC-CGC-                                          ATT                                                                     2     GCC-TGC-TCC-ATG-GTT-ATG-TTT-AGC-CGC-                                          ATT                                                                     3     GCA-GCT-GCC-TGC-CTC-ATT-TTT-AGA-GTT-                                          AGC-CGC-ATT-CAG-C                                                       7     GCT-GCC-TGC-TTC-ATG-GTT                                                 8     GCT-GCC-TGC-TTC-ATG-CTT-AGA-ATT-AGC-                                          CGC-A                                                                   9     GCT-GCC-TGC-TGC-ATG-GTT-AGC-ATT-AGC-C                                   11    GCT-GCC-TGC-TCC-CAT-GGT-TAG-GAA-TTA-                                          GCC                                                                     12    GCT-GCC-TGC-TAT-TAG-CCG                                                 13    CCT-GCT-CCA-GGG-TTA-GG                                                  14    CCT-GCT-CCG-TGG-TTA-GG                                                  ______________________________________                                    

The sequence from base 470 to 484 of the genomic RNA of poliovirus type3 Leon strain and of mutants derived from this strain is shown in Table2 below. Mutations are shown by lower case letters. The viability of thestrains is also shown, "+" meaning viable and "-" meaning not. Of theviable strains, the parental Leon strain and mutant 14 are virulent.Mutants 7, 8 and 13 are attenuated.

                  TABLE 2                                                         ______________________________________                                        Mutant    Sequence (470-484)                                                                              Viability                                         ______________________________________                                        (Leon     AUCCUAACCAUGGAG   .sup. +)                                          1         AauaUAACCAUGGAG   -                                                 2         AaCaUAACCAUGGAG   -                                                 3         AcuCUAAaaAUGagG   -                                                 7         AUCCUAACCAUGaAG   +                                                 8         AUuCUAACCAUGaAG   +                                                 9         AUgCUAACCAUGcAG   -                                                 11        AUuCcUAACCAUgGGAG -                                                 12        AUAG              -                                                 13        AUCCUAACCcUGGAG   +                                                 14        AUCCUAACCAcGGAG   +                                                 ______________________________________                                    

Mutant 7 (Table 2) was constructed by hybridising oligonucleotide 7 inTable 1 to the single stranded cloned DNA fragment in M13 phage. Thisoligonucleotide is complementary to the region 475-492 except at theposition to be mutated, where the base complementary to the desiredmutation is incorporated. In other words, oligonucleotide 7 contains Uinstead of C at the position complementary to base 482. The hybridisedM13 and oligonucleotide DNA were incubated in a reaction mixturecontaining DNA precursors and the enzymes DNA polymerase and DNA ligase.After incubation for one hour at 37° C., closed circular DNA wasisolated from this mixture by agarose gel electrophosesis. This DNA wasthen used to transform E coli muts or mutt (deficient in DNA mismatchrepair) which were then plated out on a lawn of E coli JM101.

M13 plaques which arose on this lawn of E coli were picked andpropagated and single stranded M13 phage DNA isolated. The DNAs werethen sequenced using the method of Sanger and those with the desiredmutation were identified. From these, batches of replicative form doublestranded DNA were prepared and the HindIII-Sstl fragment containing 751base pairs of infectious poliovirus cDNA, which incorporates themutation, was recovered.

The mutated cDNA fragment was then reintroduced into a derivative ofpVN23 which had been modified to include a T7 promoter. Live virus wasrecovered from the mutated full length cDNA by the production of apositive sense RNA transcript from the T7 promoter in vitro (Van derWerf et al) which was applied to Hela cells in tissue culture usingstandard techniques (Koch). After 4 to 6 days incubation a cytopathiceffect was observed and virus could be recovered from the supernatant.

Recovered virus was plaque purified and propagated in Hela cells. Thisvirus pool was used for the preparation of RNA on which the sequence ofthe virus mutant was verified using primer extension nucleotidesequencing. A portion of the pool was also used to assay neurovirulenceusing techniques described previously. The LD50 for mutant 7 was 7.5×10⁶pfu (compared to >2×10⁷ pfu for the vaccine derivative and <6×10² pfufor the neurovirulent progenitor derivative), indicating that thismutation has a definite attenuating effect on the virus.

This procedure was repeated for oligonucleotides 1 to 3, 9 and 11 to 14.Testing of virus recovered from mutant cDNA 13 (i.e. A to C at 479)revealed that this mutation also had a definite attenuating effect onvirulence (LD50=9.1×10⁶). Conversely, testing of virus recovered frommutant cDNA 14 revealed that this mutation (i.e. U to C at 480) hadlittle or no effect on neurovirulence (LD50=<7×10⁴). Attempts to recovervirus from mutant cDNAs 1, 2, 3, 9, 11 and 12 resulted in failure,indicating that they contain mutation(s) lethal for the virus.

The procedure was also repeated for oligonucleotide 8. Mutant cDNA 8 wasconstructed which contained both the mutations which are separatelyattenuating in the vaccine strain (i.e. C to U at 472) and in mutant 7(i.e. G to A at 482). Virus recovered from this double mutant cDNA wastested for neurovirulence and found to have an LD50 of >4.8×10⁷ pfu.This double mutant is therefore more attenuated than either the vaccinestrain or mutant 7.

We claim:
 1. An attenuated poliovirus having an attenuating nutation atleast at a position which is, or corresponds with, position 479 and/or482 of the genome of poliovirus type 3 Leon strain.
 2. An attenuatedvirus according to claim 1, which is a type 1 poliovirus.
 3. Anattenuated virus according to claim 1, which is a type 2 poliovirus. 4.An attenuated virus according to claim 1, which is a type 3 poliovirus.5. An attenuated virus according to claim 1, in which the base atposition 479 or at a said corresponding position is cytosine.
 6. Anattenuated virus according to claim 1, in which the base at position 482or at a said corresponding position is adenine.
 7. An attenuated virusaccording to claim 1, which also has an attenuating nutation at aposition which is, or which corresponds with, position 472 of poliovirustype 3 Leon strain.
 8. An attenuated virus according to claim 7, whichis a poliovirus which has the base U at position 472 or at a saidcorresponding position.
 9. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and an attenuated virusas claimed claim
 1. 10. A method of vaccinating a patient against apoliovirus, which method comprises administering thereto an effectiveamount of an attenuated virus as claimed in claim 1.